Method of activating regulatory T cells with alpha-2B adrenergic receptor agonists

ABSTRACT

Disclosed herein is a method of upregulating regulatory T-cells, and treating diseases that would benefit from such upregulation, by administering an alpha-2 receptor agonist.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.13/207,801, filed Aug. 11, 2011, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/374,124, filed Aug. 16, 2010,both of which are incorporated herein by reference in their entirety.

BACKGROUND

Disclosed herein is the discovery that there are alpha-2B adrenergicreceptors on a subtype of T cells, and that alpha-2 receptor agonistsmay be used to modulate the activity of such T cells and thereby treatthose diseases in which T cell dysfunction plays a role, includingneuritis, Guillan-Barre syndrome, rheumatoid arthritis, type I diabetes,multiple sclerosis (MS), graft-versus-host disease (GVHD), autoimmuneuveitis, ocular inflammation, keratoconjunctivitis sicca (dry eyesyndrome), sjogren's syndrome, atopic dermatitis, psoriasis,inflammatory bowel disease, irritable bowel syndrome, asthma, andaplastic anemia.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of upregulating regulatory Tcell function in a patient, the method comprising administering to apatient needing such upregulation an alpha-2B receptor agonist.

In another embodiment, the present invention provides a method ofupregulating regulatory T cell function in a patient, the methodcomprising administering to a patient needing such upregulation analpha-2 receptor agonist lacking significant alpha-2A receptor agonistactivity.

In another embodiment, the regulatory T cell referred to in thepreceding two paragraphs is a CD25+, FoxP3+ T cell.

In another embodiment, the present invention provides a method oftreating a disease selected from neuritis, Guillan-Barre syndrome,rheumatoid arthritis, type I diabetes, multiple sclerosis,graft-versus-host disease, autoimmune uveitis, ocular inflammation, dryeye disease, atopic dermatitis, psoriasis, inflammatory bowel disease,asthma, and aplastic anemia by administering to a patient in need ofsuch treatment an alpha-2B receptor agonist.

In another embodiment, the present invention provides a method oftreating a disease selected from neuritis, Guillan-Barre syndrome,rheumatoid arthritis, type I diabetes, multiple sclerosis,graft-versus-host disease, autoimmune uveitis, ocular inflammation, dryeye disease, atopic dermatitis, psoriasis, inflammatory bowel disease,asthma, and aplastic anemia by administering to a patient in need ofsuch treatment an alpha-2 receptor agonist lacking significant alpha-2Areceptor agonist activity.

In another embodiment, the alpha-2 receptor antagonist of the presentinvention is administered for an initial period, and then administeredagain for a second period after a withdrawal period has elapsed.

In another embodiment, the alpha-2 receptor antagonist of the presentinvention is administered for an initial period, and then administeredagain for a second period after a withdrawal period has elapsed, whereinthe initial, second, and withdrawal periods are one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, orfourteen days, or one, two, three, or four weeks.

In another embodiment, the alpha-2 receptor antagonist of the presentinvention is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2 agonist is selected from the groupconsisting of thiourea, imidazole, imidazoline, oxazole and oxazoline;or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2 agonist is selected from the groupconsisting of thiourea, imidazole, imidazoline, and oxazoline; or apharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2 agonist thiourea is a compound of theformula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2-agonist imidazoline is a compound ofthe formula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2-agonist imidazole is a compoundselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2-agonist oxazoline is a compound ofthe formula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the alpha-2-agonist of the present invention is acompound of Formula I

or a pharmaceutically acceptable salt thereof, wherein:R and R¹ are independently halogen or alkyl;R² is H or alkyl which may be unsubstituted or substituted with aheteroaryl or aryl; andHet is a heterocyclyl group selected from the group consisting ofimidazolinyl and oxazolinyl.

In another embodiment, in Formula I:

R and R¹ are independently halogen or methyl; and

R² alkyl is methyl which is substituted with a heteroaryl that ispyridyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Expression of α_(2B)-receptor but not α_(2A)-receptor inhuman T cell subsets via qPCR.

FIG. 2 shows Sustained increase in frequency of spleen CD4+ T cells thatare CD4+/CD25+ Treg cells by Compound B in the SNL Rat Model ofAllodynic Neuropathic Pain.

FIG. 3 shows that α_(2B)-receptor agonist Compound B is analgesic inestablished MS pain and shows modulation of Treg cells.

FIG. 4 shows that Compound B has a significant effect on clinical courseof disease in proteolipid-induced model of relapsing remitting EAE.

FIG. 5 shows that Compound B significantly reduced the presence ofimmune cells in the CNS in PLP-induced model of relapsing remitting EAE.

FIG. 6 shows that Compound C has a significant effect on clinical courseof disease in proteolipid-induced model of relapsing remitting EAE.

FIG. 7 shows that Compound C significantly increases the frequency ofregulatory T cells in the spinal cord in PLP-induced model of relapsingremitting EAE.

FIG. 8 shows that Compound B has a significant effect on clinicaldisease and on the number of inflammatory cells in the aqueous humor ofEAAU rats.

FIG. 9 shows that Compound B has a significant effect on proteinconcentration in the aqueous humor of EIU rats.

FIG. 10 shows that Compound B normalizes the levels of blood neutrophilsand lymphocytes in the rat EIU model.

FIG. 11 shows that Treatment of donor with Compound B starting atinitiation of desiccating stress significantly reduced recipient tearTNF-alpha and IL17 levels.

FIG. 12 shows that Treatment with Compound C in a therapeutic modestarting after exposure of mice to desiccating stress significantlyreduced goblet cell loss and T cell infiltration into the conjunctiva.

DETAILED DESCRIPTION OF THE INVENTION

Alpha-2 Receptor Agonists

Alpha-2 receptor agonists are those compounds that activate alpha-2adrenergic receptors. There are three subtypes of this receptor,designated A, B, and C. A compound is an “alpha-2B receptor agonist” ifit has greater than 25% efficacy relative to brimonidine at the alpha-2Badrenergic receptor; a compound is an “alpha-2C receptor agonist” if ithas greater than 25% efficacy relative to brimonidine at the alpha-2Cadrenergic receptor; and a compound is an “alpha-2B/2C receptor agonist”if it has greater than 25% efficacy relative to brimonidine at both thealpha-2B and alpha-2C adrenergic receptors. The definitions are notmutually exclusive: a compound that is an alpha-2B receptor agonist canalso be an alpha-2B/2C receptor agonist; and compound that is analpha-2C receptor agonist can also be an alpha-2B/2C receptor agonist.

In one embodiment, the methods of the present invention use alpha-2agonists lacking significant activity at the alpha-2A receptor subtype.An agonist lacks significant alpha-2A receptor activity if the agonisthas less than 40% of the efficacy of brimonidine at the alpha-2Areceptor subtype. Compounds of the invention include, therefore,alpha-2B receptor agonists; alpha-2B receptor agonists lackingsignificant alpha-2A activity; alpha-2C receptor agonists; alpha-2Creceptor agonists lacking significant alpha-2A activity; alpha 2B/2Creceptor agonists; and alpha 2B/2C receptor agonists lacking significantalpha-2A activity. Any of the foregoing compounds may be used, even ifthey bind receptors other than alpha-2 receptors; for example, alpha-1receptor agonists may be used, provided that the alpha-1 agonists alsohave greater than 25% efficacy relative to brimonidine at one or both ofthe alpha-2B and alpha-2C receptor subtypes, and lack significantalpha-2A receptor activity.

Efficacy, also known as intrinsic activity, is a measure of maximalreceptor activation achieved by a compound and can be determined usingany accepted assay of alpha-adrenergic receptor activation, such as acAMP or Receptor Selection and Amplification Technology (RSAT). Efficacyis represented as a ratio or percentage of the maximal effect of thedrug to the maximal effect of a standard agonist for each receptorsubtype. Brimonidine, itself an alpha-2B receptor agonist (it is has100% the efficacy of brimonidine at the alpha-2B adrenergic receptor),is used as the standard agonist for the alpha-2B adrenergic receptors.

Agonist activity can be characterized using any of a variety of routineassays, including, for example, Receptor Selection and AmplificationTechnology (RSAT) assays (Messier et al., Pharmacol. Toxicol. 76:308-11(1995); cyclic AMP assays (Shimizu et al., J. Neurochem. 16:1609-1619(1969)); and cytosensor microphysiometry assays (Neve et al., J. Biol.Chem. 267:25748-25753 (1992)). Such assays generally are performed usingcells that naturally express only a single alpha-adrenergic receptorsubtype, or using transfected cells expressing a single recombinantalpha-adrenergic receptor subtype. The adrenergic receptor can be ahuman receptor or homolog of a human receptor having a similarpharmacology.

The RSAT assay measures receptor-mediated loss of contact inhibitionresulting in selective proliferation of receptor-containing cells in amixed population of confluent cells. The increase in cell number isassessed with an appropriate detectable marker gene such asbeta-galactosidase, if desired, in a high throughput or ultra highthroughput assay format. Receptors that activate the G protein, Gq,elicit the proliferative response. Alpha-adrenergic receptors, whichnormally couple to Gi, activate the RSAT response when coexpressed witha hybrid Gq protein containing a Gi receptor recognition domain,designated Gq/i5. Conklin et al., Nature 363:274-6 (1993)).

As an example, an RSAT assay can be performed essentially as follows.NIH-3T3 cells are plated at a density of 2×10⁶ cells in 15 cm dishes andmaintained in Dulbecco's modified Eagle's medium supplemented with 10%calf serum. One day later, cells are cotransfected by calcium phosphateprecipitation with mammalian expression plasmids encodingp-SV-β-galactosidase (5-10 μg), receptor (1-2 μg) and protein (1-2 μg).Carrier DNA, for example 40 μg salmon sperm DNA, also can be included toincrease transfection efficiency. Fresh media is added on the followingday; one to two days later, cells are harvested and frozen in 50 assayaliquots. Transfected cells are thawed, and 100 μl of cells added to 100μl aliquots of compound to be tested, with various concentrationsassayed in triplicate, for example, in 96-well plates. Incubationcontinues for 72 to 96 hours at 37° C. After washing withphosphate-buffered saline, β-galactosidase activity is determined byadding 200 μl of chromogenic substrate (3.5 mMO-nitrophenyl-β-D-galactopyranoside/0.5% NP-40 in phosphate bufferedsaline), incubating overnight at 30° C., and measuring optical densityat 420 nm. The absorbency is a measure of enzyme activity, which dependson cell number and reflects receptor-mediated cell proliferation. TheEC₅₀ and maximal effect (i.e., efficacy) of each drug at each receptoris determined.

Alpha-2B and -2C receptor agonists, including those lacking significantalpha-2A receptor activity, are known in the art. Detailed informationregarding alpha-2 agonists, including their structure, synthesis, andactivity, may be found in U.S. Pat. No. 6,329,369, No. 6,534,542, No.6,545,182, No. 6,787,517, No. 6,841,684, and No. 7,091,232; in U.S.Patent Application Publication No. 2003/0092766, No. 2004/0132824, No.2004/0220402, No. 2005/0075366, and No. 2005/0267186; and in U.S. patentapplication Ser. No. 11/172,229, No. 11/232,323, No. 11/232,341, No.60/613,870, No. 60/695,650, No. 60/747,444, No. 60/884,718, No.60/917,828, No. 60/911,422, No. 60/911,478, and No. 60/948,389, thedisclosures of all which are incorporated herein by reference.

One can use in the methods of the invention any pharmaceuticallyacceptable salt, prodrug, isomer, or racemate of any alpha-2 receptoragonist.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched and comprising about 1 to about 20 carbon atoms in the chain.Preferred alkyl groups contain about 1 to about 12 carbon atoms in thechain. More preferred alkyl groups contain about 1 to about 6 carbonatoms in the chain. Branched means that one or more lower alkyl groupssuch as methyl, ethyl or propyl, are attached to a linear alkyl chain.“Lower alkyl” means a group having about 1 to about 6 carbon atoms inthe chain which may be straight or branched. “Alkyl” may beunsubstituted or optionally substituted by one or more substituentswhich may be the same or different, each substituent being independentlyselected from the group consisting of halo, alkyl, aryl, heterocyclyl,heteroaryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, oxime(e.g., ═N—OH), —NH(alkyl), —NH(cycloalkyl), —N(alkyl)₂, —O—C(O)-alkyl,—O—C(O)-aryl, —O—C(O)-cycloalkyl, —SF₅, carboxy and —C(O)O-alkyl.Non-limiting examples of suitable alkyl groups include methyl, ethyl,n-propyl, isopropyl and t-butyl.

“Alkenyl” means an aliphatic hydrocarbon group containing at least onecarbon-carbon double bond and which may be straight or branched andcomprising about 2 to about 15 carbon atoms in the chain. Preferredalkenyl groups have about 2 to about 12 carbon atoms in the chain; andmore preferably about 2 to about 6 carbon atoms in the chain. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl, are attached to a linear alkenyl chain. “Lower alkenyl” meansabout 2 to about 6 carbon atoms in the chain which may be straight orbranched. “Alkenyl” may be unsubstituted or optionally substituted byone or more substituents which may be the same or different, eachsubstituent being independently selected from the group consisting ofhalo, alkyl, aryl, cycloalkyl, cyano, alkoxy and —S(alkyl). Non-limitingexamples of suitable alkenyl groups include ethenyl, propenyl,n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl and decenyl.

“Alkylene” means a difunctional group obtained by removal of a hydrogenatom from an alkyl group that is defined above. Non-limiting examples ofalkylene include methylene, ethylene and propylene.

“Alkynyl” means an aliphatic hydrocarbon group containing at least onecarbon-carbon triple bond and which may be straight or branched andcomprising about 2 to about 15 carbon atoms in the chain. Preferredalkynyl groups have about 2 to about 12 carbon atoms in the chain; andmore preferably about 2 to about 4 carbon atoms in the chain. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl, are attached to a linear alkynyl chain. “Lower alkynyl” meansabout 2 to about 6 carbon atoms in the chain which may be straight orbranched. Non-limiting examples of suitable alkynyl groups includeethynyl, propynyl, 2-butynyl and 3-methylbutynyl. “Alkynyl” may beunsubstituted or optionally substituted by one or more substituentswhich may be the same or different, each substituent being independentlyselected from the group consisting of alkyl, aryl and cycloalkyl.

“Aryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 6 to about 14 carbon atoms, preferably about 6 to about10 carbon atoms. The aryl group can be optionally substituted with oneor more “ring system substituents” which may be the same or different,and are as defined herein. Non-limiting examples of suitable aryl groupsinclude phenyl and naphthyl.

“Heteroaryl” means an aromatic monocyclic or multicyclic ring systemcomprising about 5 to about 14 ring atoms, preferably about 5 to about10 ring atoms, in which one or more of the ring atoms is an elementother than carbon, for example nitrogen, oxygen or sulfur, alone or incombination. Preferred heteroaryls contain about 5 to about 6 ringatoms. The “heteroaryl” can be optionally substituted by one or more“ring system substituents” which may be the same or different, and areas defined herein. The prefix aza, oxa or thia before the heteroarylroot name means that at least a nitrogen, oxygen or sulfur atomrespectively, is present as a ring atom. A nitrogen atom of a heteroarylcan be optionally oxidized to the corresponding N-oxide. “Heteroaryl”may also include a heteroaryl as defined above fused to an aryl asdefined above. Non-limiting examples of suitable heteroaryls includepyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (includingN-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl,pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl,1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl,oxindolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl,benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl,quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl,pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl,1,2,4-triazinyl, benzothiazolyl and the like. The term “heteroaryl” alsorefers to partially saturated heteroaryl moieties such as, for example,tetrahydroisoquinolyl, tetrahydroquinolyl and the like.

“Aralkyl” or “arylalkyl” means an aryl-alkyl- group in which the aryland alkyl are as previously described. Preferred aralkyls comprise alower alkyl group. Non-limiting examples of suitable aralkyl groupsinclude benzyl, 2-phenethyl and naphthalenylmethyl. The bond to theparent moiety is through the alkyl.

“Alkylaryl” means an alkyl-aryl- group in which the alkyl and aryl areas previously described. Preferred alkylaryls comprise a lower alkylgroup. Non-limiting example of a suitable alkylaryl group is tolyl. Thebond to the parent moiety is through the aryl.

“Cycloalkyl” means a non-aromatic mono- or multicyclic ring systemcomprising about 3 to about 10 carbon atoms, preferably about 5 to about10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7ring atoms. The cycloalkyl can be optionally substituted with one ormore “ring system substituents” which may be the same or different, andare as defined above. Non-limiting examples of suitable monocycliccycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyland the like. Non-limiting examples of suitable multicyclic cycloalkylsinclude 1-decalinyl, norbornyl, adamantyl and the like.

“Cycloalkylalkyl” means a cycloalkyl moiety as defined above linked viaan alkyl moiety (defined above) to a parent core. Non-limiting examplesof suitable cycloalkylalkyls include cyclohexylmethyl, adamantylmethyland the like.

“Cycloalkenyl” means a non-aromatic mono or multicyclic ring systemcomprising about 3 to about 10 carbon atoms, preferably about 5 to about10 carbon atoms which contains at least one carbon-carbon double bond.Preferred cycloalkenyl rings contain about 5 to about 7 ring atoms. Thecycloalkenyl can be optionally substituted with one or more “ring systemsubstituents” which may be the same or different, and are as definedabove. Non-limiting examples of suitable monocyclic cycloalkenylsinclude cyclopentenyl, cyclohexenyl, cyclohepta-1,3-dienyl, and thelike. Non-limiting example of a suitable multicyclic cycloalkenyl isnorbornylenyl.

“Cycloalkenylalkyl” means a cycloalkenyl moiety as defined above linkedvia an alkyl moiety (defined above) to a parent core. Non-limitingexamples of suitable cycloalkenylalkyls include cyclopentenylmethyl,cyclohexenylmethyl and the like.

“Halogen” means fluorine, chlorine, bromine, or iodine. Preferred arefluorine, chlorine and bromine.

“Ring system substituent” means a substituent attached to an aromatic ornon-aromatic ring system which, for example, replaces an availablehydrogen on the ring system. Ring system substituents may be the same ordifferent, each being independently selected from the group consistingof alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl,heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl,hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo,nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl,alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,cycloalkyl, heterocyclyl, —SF₅, —OSF₅ (for aryl), —O—C(O)-alkyl,—O—C(O)-aryl, —O—C(O)-cycloalkyl, —C(═N—CN)—NH₂, —C(═NH)—NH₂,—C(═NH)—NH(alkyl), oxime (e.g., ═N—OH), —NY₁Y₂, -alkyl-NY₁Y₂,—C(O)NY₁Y₂, —SO₂NY₁Y₂ and —SO₂NY₁Y₂, wherein Y₁ and Y₂ can be the sameor different and are independently selected from the group consisting ofhydrogen, alkyl, aryl, cycloalkyl, and aralkyl. “Ring systemsubstituent” may also mean a single moiety which simultaneously replacestwo available hydrogens on two adjacent carbon atoms (one H on eachcarbon) on a ring system. Examples of such moiety are methylene dioxy,ethylenedioxy, —C(CH₃)₂— and the like which form moieties such as, forexample:

“Heteroarylalkyl” means a heteroaryl moiety as defined above linked viaan alkyl moiety (defined above) to a parent core. Non-limiting examplesof suitable heteroaryls include 2-pyridinylmethyl, quinolinylmethyl andthe like.

“Heterocyclyl” means a non-aromatic saturated monocyclic or multicyclicring system comprising about 3 to about 10 ring atoms, preferably about5 to about 10 ring atoms, in which one or more of the atoms in the ringsystem is an element other than carbon, for example nitrogen, oxygen orsulfur, alone or in combination. There are no adjacent oxygen and/orsulfur atoms present in the ring system. Preferred heterocyclyls containabout 5 to about 6 ring atoms. The prefix aza, oxa or thia before theheterocyclyl root name means that at least a nitrogen, oxygen or sulfuratom respectively is present as a ring atom. Any —NH in a heterocyclylring may exist protected such as, for example, as an —N(Boc), —N(CBz),—N(Tos) group and the like; such protections are also considered part ofthis invention. The heterocyclyl can be optionally substituted by one ormore “ring system substituents” which may be the same or different, andare as defined herein. The nitrogen or sulfur atom of the heterocyclylcan be optionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclylrings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl,thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl,tetrahydrothiophenyl, lactam, lactone, and the like. “Heterocyclyl” alsoincludes heterocyclyl rings as described above wherein ═O replaces twoavailable hydrogens on the same ring carbon atom. Example of such moietyis pyrrolidone:

“Heterocyclylalkyl” means a heterocyclyl moiety as defined above linkedvia an alkyl moiety (defined above) to a parent core. Non-limitingexamples of suitable heterocyclylalkyls include piperidinylmethyl,piperazinylmethyl and the like.

“Heterocyclenyl” means a non-aromatic monocyclic or multicyclic ringsystem comprising about 3 to about 10 ring atoms, preferably about 5 toabout 10 ring atoms, in which one or more of the atoms in the ringsystem is an element other than carbon, for example nitrogen, oxygen orsulfur atom, alone or in combination, and which contains at least onecarbon-carbon double bond or carbon-nitrogen double bond. There are noadjacent oxygen and/or sulfur atoms present in the ring system.Preferred heterocyclenyl rings contain about 5 to about 6 ring atoms.The prefix aza, oxa or thia before the heterocyclenyl root name meansthat at least a nitrogen, oxygen or sulfur atom respectively is presentas a ring atom. The heterocyclenyl can be optionally substituted by oneor more ring system substituents, wherein “ring system substituent” isas defined above. The nitrogen or sulfur atom of the heterocyclenyl canbe optionally oxidized to the corresponding N-oxide, S-oxide orS,S-dioxide. Non-limiting examples of suitable heterocyclenyl groupsinclude 1,2,3,4-tetrahydropyridinyl, 1,2-dihydropyridinyl,1,4-dihydropyridinyl, 1,2,3,6-tetrahydropyridinyl,1,4,5,6-tetrahydropyrimidinyl, 2-pyrrolinyl, 3-pyrrolinyl,2-imidazolinyl, 2-pyrazolinyl, dihydroimidazolyl, dihydrooxazolyl,dihydrooxadiazolyl, dihydrothiazolyl, 3,4-dihydro-2H-pyranyl,dihydrofuranyl, fluorodihydrofuranyl, 7-oxabicyclo[2.2.1]heptenyl,dihydrothiophenyl, dihydrothiopyranyl, and the like. “Heterocyclenyl”also includes heterocyclenyl rings as described above wherein ═Oreplaces two available hydrogens on the same ring carbon atom. Exampleof such moiety is pyrrolidinone:

“Heterocyclenylalkyl” means a heterocyclenyl moiety as defined abovelinked via an alkyl moiety (defined above) to a parent core.

It should be noted that in hetero-atom containing ring systems of thisinvention, there are no hydroxyl groups on carbon atoms adjacent to a N,O or S, as well as there are no N or S groups on carbon adjacent toanother heteroatom. Thus, for example, in the ring:

there is no —OH attached directly to carbons marked 2 and 5.

It should also be noted that tautomeric forms such as, for example, themoieties:

are considered equivalent in certain embodiments of this invention.

“Alkynylalkyl” means an alkynyl-alkyl- group in which the alkynyl andalkyl are as previously described. Preferred alkynylalkyls contain alower alkynyl and a lower alkyl group. The bond to the parent moiety isthrough the alkyl. Non-limiting examples of suitable alkynylalkyl groupsinclude propargylmethyl.

“Heteroaralkyl” means a heteroaryl-alkyl- group in which the heteroaryland alkyl are as previously described. Preferred heteroaralkyls containa lower alkyl group. Non-limiting examples of suitable aralkyl groupsinclude pyridylmethyl, and quinolin-3-ylmethyl. The bond to the parentmoiety is through the alkyl.

“Hydroxyalkyl” means a HO-alkyl- group in which alkyl is as previouslydefined. Preferred hydroxyalkyls contain lower alkyl. Non-limitingexamples of suitable hydroxyalkyl groups include hydroxymethyl and2-hydroxyethyl.

“Acyl” means an H—C(O)—, alkyl-C(O)— or cycloalkyl-C(O)—, group in whichthe various groups are as previously described. The bond to the parentmoiety is through the carbonyl. Preferred acyls contain a lower alkyl.Non-limiting examples of suitable acyl groups include formyl, acetyl andpropanoyl.

“Aroyl” means an aryl-C(O)— group in which the aryl group is aspreviously described. The bond to the parent moiety is through thecarbonyl. Non-limiting examples of suitable groups include benzoyl and1-naphthoyl.

“Alkoxy” means an alkyl-O— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkoxy groupsinclude methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond tothe parent moiety is through the ether oxygen.

“Aryloxy” means an aryl-O— group in which the aryl group is aspreviously described. Non-limiting examples of suitable aryloxy groupsinclude phenoxy and naphthoxy. The bond to the parent moiety is throughthe ether oxygen.

“Aralkyloxy” means an aralkyl-O— group in which the aralkyl group is aspreviously described. Non-limiting examples of suitable aralkyloxygroups include benzyloxy and 1- or 2-naphthalenemethoxy. The bond to theparent moiety is through the ether oxygen.

“Alkylthio” means an alkyl-S— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkylthio groupsinclude methylthio and ethylthio. The bond to the parent moiety isthrough the sulfur.

“Arylthio” means an aryl-S— group in which the aryl group is aspreviously described. Non-limiting examples of suitable arylthio groupsinclude phenylthio and naphthylthio. The bond to the parent moiety isthrough the sulfur.

“Aralkylthio” means an aralkyl-S— group in which the aralkyl group is aspreviously described. Non-limiting example of a suitable aralkylthiogroup is benzylthio. The bond to the parent moiety is through thesulfur.

“Alkoxycarbonyl” means an alkyl-O—CO— group. Non-limiting examples ofsuitable alkoxycarbonyl groups include methoxycarbonyl andethoxycarbonyl. The bond to the parent moiety is through the carbonyl.

“Aryloxycarbonyl” means an aryl-O—C(O)— group. Non-limiting examples ofsuitable aryloxycarbonyl groups include phenoxycarbonyl andnaphthoxycarbonyl. The bond to the parent moiety is through thecarbonyl.

“Aralkoxycarbonyl” means an aralkyl-O—C(O)— group. Non-limiting exampleof a suitable aralkoxycarbonyl group is benzyloxycarbonyl. The bond tothe parent moiety is through the carbonyl.

“Alkylsulfonyl” means an alkyl-S(O₂)— group. Preferred groups are thosein which the alkyl group is lower alkyl. The bond to the parent moietyis through the sulfonyl.

“Arylsulfonyl” means an aryl-S(O₂)— group. The bond to the parent moietyis through the sulfonyl.

The term “substituted” means that one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency under the existingcircumstances is not exceeded, and that the substitution results in astable compound. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds. By“stable compound” or “stable structure” is meant a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

The term “purified”, “in purified form” or “in isolated and purifiedform” for a compound refers to the physical state of said compound afterbeing isolated from a synthetic process (e.g. from a reaction mixture),or natural source or combination thereof. Thus, the term “purified”, “inpurified form” or “in isolated and purified form” for a compound refersto the physical state of said compound after being obtained from apurification process or processes described herein or well known to theskilled artisan (e.g., chromatography, recrystallization and the like)in sufficient purity to be characterizable by standard analyticaltechniques described herein or well known to the skilled artisan.

It should also be noted that any carbon as well as heteroatom withunsatisfied valences in the text, schemes, examples and Tables herein isassumed to have the sufficient number of hydrogen atom(s) to satisfy thevalences. And any one or more of these hydrogen atoms can be deuterium.

When a functional group in a compound is termed “protected”, this meansthat the group is in modified form to preclude undesired side reactionsat the protected site when the compound is subjected to a reaction.Suitable protecting groups will be recognized by those with ordinaryskill in the art as well as by reference to standard textbooks such as,for example, T. W. Greene et al, Protective Groups in organic Synthesis(1991), Wiley, New York.

When any variable (e.g., aryl, heterocycle, R², etc.) occurs more thanone time in any constituent or in Formula I, its definition on eachoccurrence is independent of its definition at every other occurrence.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

Pharmaceutically Acceptable Salts

Alpha-2 receptor agonists may be used as their pharmaceuticallyacceptable salts.

A “pharmaceutically acceptable salt” is any salt that retains theactivity of the parent compound and does not impart any additionaldeleterious or untoward effects on the subject to which it isadministered and in the context in which it is administered compared tothe parent compound. A pharmaceutically acceptable salt also refers toany salt which may form in vivo as a result of administration of anacid, another salt, or a prodrug which is converted into an acid orsalt.

Pharmaceutically acceptable salts of acidic functional groups may bederived from organic or inorganic bases. The salt may comprise a mono orpolyvalent ion. Of particular interest are the inorganic ions lithium,sodium, potassium, calcium, and magnesium. Organic salts may be madewith amines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanol amines. Salts may also be formed with caffeine,tromethamine and similar molecules. Hydrochloric acid or some otherpharmaceutically acceptable acid may form a salt with a compound thatincludes a basic group, such as an amine or a pyridine ring.

Prodrugs

One can use in the compositions and methods of the invention a prodrugof any alpha-2 receptor agonist.

A “prodrug” is a compound which is converted to a therapeutically activecompound after administration, and the term should be interpreted asbroadly herein as is generally understood in the art. While notintending to limit the scope of the invention, conversion may occur byhydrolysis of an ester group or some other biologically labile group.Generally, but not necessarily, a prodrug is inactive or less activethan the therapeutically active compound to which it is converted. Esterprodrugs of the compounds disclosed herein are specificallycontemplated. An ester may be derived from a carboxylic acid of C1(i.e., the terminal carboxylic acid of a natural prostaglandin), or anester may be derived from a carboxylic acid functional group on anotherpart of the molecule, such as on a phenyl ring. While not intending tobe limiting, an ester may be an alkyl ester, an aryl ester, or aheteroaryl ester. In this context (definition of “prodrug”), the term“alkyl” has the meaning generally understood by those skilled in the artand refers to linear, branched, or cyclic alkyl moieties. C₁₋₆ alkylesters are particularly useful, where alkyl part of the ester has from 1to 6 carbon atoms and includes, but is not limited to, methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, t-butyl, pentylisomers, hexyl isomers, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and combinations thereof having from 1-6 carbon atoms, etc.

The alpha-2 receptor agonists of the invention may be eithersynthetically produced, or may be produced within the body afteradministration of a prodrug. Hence, the term “alpha-2 receptor agonist”encompasses both compounds produced by a manufacturing process and thosecompounds formed in vivo only when another drug administered.

Isomers and Racemates

One can use in the compositions and methods of the invention anenantiomer, stereoisomer, or other isomer of an alpha-2 receptoragonist. One can also use in the compositions and methods of theinvention a racemic mixture or one or both racemates, in any proportion.

Dose

The precise dose and frequency of administration depends on the severityand nature of the patient's condition, on the manner of administration,on the potency and pharmacodynamics of the particular compound employed,and on the judgment of the prescribing physician. Determining dose is aroutine matter that is well within the capability of someone of ordinaryskill in the art. In general, alpha-2 receptor agonists are administeredin therapeutically effective doses, that is, at a dose that issufficient to produce the desired therapeutic effect.

In one embodiment, the compounds of the invention (alpha-2B receptoragonists; alpha-2B receptor agonists lacking significant alpha-2Aactivity; alpha-2C receptor agonists; alpha-2C receptor agonists lackingsignificant alpha-2A activity; alpha 2B/2C receptor agonists; and alpha2B/2C receptor agonists lacking significant alpha-2A activity) providelong-term relief—that is, relief that endures for one or more days afterthe compounds are withdrawn. Hence, in one embodiment, the method of theinvention comprises administering to a patient a compound of theinvention for an initial period, then administering the compound againfor a second period after a withdrawal period has elapsed. The initial,second, and withdrawal periods may be one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days, orone, two, three, or four weeks, and may be the same or different. Hence,for example, one can administer a compound of the invention for threedays, and then administer the compound again for three days, no soonerthan three days after the compound was last administered; or one canadminister a compound of the invention for two weeks, and thenadminister the compound again for one week no sooner than one week afterthe compound was last administered.

In another embodiment, the initial and second periods are variable andthe withdrawal period is fixed. In such embodiments, the initial andsecond period is at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, or fourteen days, or one,two, three, or four weeks. Hence, for example, one can administer acompound of the invention for at least three days, and then administerthe compound again for at least three days no sooner than six days afterthe compound was last administered; or one can administer a compound ofthe invention for at least one week, and then administer the compoundagain for at least a week no sooner than one week after the compound waslast administered.

Excipients and Dosage Forms

Those skilled in the art will readily understand that alpha-2 receptoragonists can be admixed with pharmaceutically acceptable excipientswhich are well known in the art.

A pharmaceutical composition to be administered systemically may beconfected as a powder, pill, tablet or the like, or as a solution,emulsion, suspension, aerosol, syrup or elixir suitable for oral orparenteral administration, inhalation or topical administration to theeye or skin.

For solid dosage forms or medicaments, non-toxic solid carriers include,but are not limited to, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, the polyalkylene glycols,talcum, cellulose, glucose, sucrose and magnesium carbonate. The soliddosage forms may be uncoated or they may be coated by known techniquesto delay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They may also be coated by the technique described inU.S. Pat. No. 4,256,108, U.S. Pat. No. 4,166,452, and U.S. Pat. No.4,265,874 to form osmotic therapeutic tablets for control release.Liquid pharmaceutically administrable dosage forms can, for example,comprise a solution or suspension of one or more of the presently usefulcompounds and optional pharmaceutical adjutants in a carrier, such asfor example, water, saline, aqueous dextrose, glycerol, ethanol and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like. Typical examples of suchauxiliary agents are sodium acetate, sorbitan monolaurate,triethanolamine, sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 16thEdition, 1980. The composition of the formulation to be administered, inany event, contains a quantity of one or more of the presently usefulcompounds in an amount effective to provide the desired therapeuticeffect.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol and the like. Inaddition, if desired, the injectable pharmaceutical compositions to beadministered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like.

Activating T Cells

T cells are a class of lymphocytes having specific T cell receptors(TCRs) that are produced as a result of gene rearrangement. T cells havediverse roles, which are accomplished by the differentiation of distinctsubsets of T cells, recognizable by discrete patterns of geneexpression. Several major T cell subsets are recognized based onreceptor expression, such as TCR-α/β, and TCRγ/δ and invariant naturalkiller cells. Other T cell subsets are defined by the surface moleculesand cytokines secreted therefrom. For example, T helper cells (CD4cells) secrete cytokines, and help B cells and cytotoxic T cells tosurvive and carry out effector functions. Cytotoxic T cells (CTLs) aregenerally CD8 cells, and they are specialized to kill target cells, suchas infected cells or tumor cells. Natural killer (NK) cells are relatedto T cells, but do not have TCRs, and have a shorter lifespan, althoughthey do share some functions with T cells and are able to secretecytokines and kill some kinds of target cells.

Human and mouse peripheral blood contains a small population of T celllymphocytes that express the T regulatory phenotype (“Treg”), i.e.,positive for both CD4 and CD25 antigens (i.e., those CD4⁺ T cells thatare also distinctly positive for CD25). First characterized in mice,where they constitute 6-10% of lymph node and splenic CD4⁺ T cellpopulations, this population of CD4⁺CD25⁺ cells represents approximatelyonly 5-10% of human peripheral blood mononuclear cells (PBMC), or 2-7%of CD4⁺ T cells, although some donors exhibit a more distinct populationof CD4⁺ and CD25⁺ cells. About 1-2% of human peripheral blood PBMCs areboth CD4 positive (CD4⁺) and CD25 brightly positive (CD25⁺) cells.

There are several subsets of Treg cells (Bluestone et al., Nature Rev.Immunol., 3:253 (2003)). One subset of regulatory cells develops in thethymus. Thymic derived Treg cells function by a cytokine-independentmechanism, which involves cell to cell contact (Shevach, Nature Rev.Immunol. 2:389 (2002)). They are essential for the induction andmaintenance of self-tolerance and for the prevention of autoimmunity(Shevach, Annu. Rev. Immunol. 18:423-449 (2000); Stephens et al., 2001;Taams et al., 2001; Thornton et al., 1998; Salomon et al., Immunity12:431-440 (2000); Sakaguchi et al., Immunol. Rev. 182:18-32 (2001)).These professional regulatory cells prevent the activation andproliferation of autoreactive T cells that have escaped thymic deletionor recognize extrathymic antigens, thus they are critical forhomeostasis and immune regulation, as well as for protecting the hostagainst the development of autoimmunity (Suri-Payer et al., J. Immunol.157:1799-1805 (1996); Asano et al., J. Exp. Med. 184:387-396 (1996);Bonomo et al., J. Immunol. 154:6602-6611 (1995); Willerford et al.,Immunity 3:521-530 (1995); Takahashi et al., Int. Immunol. 10:1969-1980(1998); Salomon et al., Immunity 12:431-440 (2000); Read et al., J. Exp.Med. 192:295-302 (2000). Thus, immune regulatory CD4⁺CD25⁺ T cells areoften referred to as “professional suppressor cells.”

However, Treg cells can also be generated by the activation of mature,peripheral CD4⁺ T cells. Studies have indicated that peripherallyderived Treg cells mediate their inhibitory activities by producingimmunosuppressive cytokines, such as transforming growth factor-beta(TGF-β) and IL-10 (Kingsley et al., J. Immunol. 168:1080 (2002);Nakamura et al., J. Exp. Med. 194:629-644 (2001)). Afterantigen-specific activation, these Treg cells can non-specificallysuppress proliferation of either CD4⁺ or CD25⁺ T cells (demonstrated byFACS sorting in low dose immobilized anti-CD3 mAb-based co-culturesuppressor assays by Baecher-Allan et al., J. Immunol. 167(3):1245-1253(2001)).

Recently, Riley et al. (“Human T regulatory cell therapy: take a billionor so and call me in the morning,” Immunity, 30(5), 656-665 (2009)) haveshown that regulatory T cells are critical in several pathologicalconditions involving immune activation (Riley et al., 2009). CD25+,FoxP3+ Tregs have the capacity to block immune responses inflammationand tissue destruction by suppressing the functions of an array of celltypes including conventional CD4+ helper T cells, B cell antibodyproduction, CD8+ cytotoxic activity and antigen-presenting cell functionand maturation (Tang & Bluestone, 2008). A

diminished frequency or dysfunction of Tregs has been reported in manyhuman diseases (Tran & Shevach, 2009).

In one embodiment, the method of the invention comprises administeringan alpha-2 agonist to upregulate regulatory T cell function in a patientin whom such upregulation would be beneficial. In another embodiment,the regulatory T cell is a CD25+, FoxP3+ T cell. In another embodiment,the method of the invention comprises administering an alpha-2 agonistto treat diseases such as neuritis, Guillan-Barre syndrome, rheumatoidarthritis, type I diabetes, multiple sclerosis (MS), graft-versus-hostdisease (GVHD), autoimmune uveitis, ocular inflammation, dry eyedisease, atopic dermatitis, psoriasis, inflammatory bowel disease,asthma, and aplastic anemia.

EXAMPLES

The invention is illustrated by the following examples. This is providedfor illustration only; many more embodiments are possible.

Evidence for Alpha-28 Action on Regulatory T Cells

The inventors have tested whether α_(2B)-selective compounds regulateneuropathic pain states via immune-related mechanisms. Treatment withthe α_(2B) agonist, Compound A, attenuated the spinal nerve ligationsurgery-induced increase of IL-2 levels (Table 1). IL-2 is apro-inflammatory cytokine which is essential for regulating T-lymphocyteproliferation. This finding indicates that the α_(2B) agonist effect onacute and chronic allodynia reversal (as described in U.S. Pat. No.7,345,065, the disclosure of which is incorporated herein by reference)might be mediated via immune cells, particularly T cells. The inventorshave also observed expression of α_(2B) receptor, but not α_(2A)receptor, in different subtypes of human T lymphocytes via qPCR. To theinventors' knowledge this is the first demonstration of expression ofα_(2B)-receptor subtype in T cells (FIG. 1). Additional evidence for therole of immune cells in the persistent pain alleviation mechanismsmediated by α_(2B)-receptor agonists was obtained by the fact that theanalgesic effect of a second α_(2B) agonist with a different structure,Compound B, was blockable with the immunosuppressant drug FK506 (Table2). This finding suggests that α_(2B)-receptor-induced long-termallodynia reversal requires the presence of activated lymphocytes, sinceFK506 has been shown to be a potent blocker of T-lymphocyte activation(Small et al, 1996). This finding was confirmed with yet another α_(2B)agonist with a different structure, Compound C, where in FK506 was ableto block the long-term analgesic activity of Compound C in the spinalnerve ligation model (Table 3).

Further, the inventors have explored the subtype of T cells involved inα_(2B)-receptor-mediated effects and observe a significant andpersistent increase in the number of regulatory T Cells (Tregs) in thelymphatic organs in animals with neuropathic pain that have been treatedwith α_(2B)-receptor agonists (FIG. 2). The inventors observed no effectof compound on Treg cell numbers in naïve animals. This suggests thatthe effect of α_(2B)-receptor agonists on Treg cells is dependent onantigen stimulation and there may be antigen-selective expansion of Treg cells.

Effect of Alpha-2B Agonists in a Model of Multiple Sclerosis

The inventors have demonstrated that this mechanism of α_(2B)-receptoragonists is generalizable to a second model of pathological T cellactivation, a model of multiple sclerosis, and a second species. In miceimmunized with proteolipid protein, which causes a relapsing-remittingform of experimental autoimmune encephalomyelitis, Compound B (3mg/kg/day by osmotic minipump) selectively boosted Treg number andreduced pain [FIG. 3].

TABLE 1 Attenuation of levels of IL-2 in various tissues from SNL ratstreated with Compound A (2.4 mg/kg/day via osmotic minipump) or withvehicle Ipsilateral Ipsilateral Ipsilateral DRGL4 DRGL5, L6 spinal cordSerum Naïve  68 ± 8.5 138 ± 0.88 397.33 ± 44.58   233.33 ± 56.25 SNLrats 139.33 ± 15.76 586 ± 7.21 523 ± 44.80  768.33 ± 271.94 Vehicle SNLrats 101.63 ± 20.55   211 ± 33.72** 345 ± 19.34   978 ± 77.13 Compound A24 hours SNL rats  69.52 ± 9.42**  138.35 ± 22.59** 351.75 ± 17.19* 380.25 ± 61.67 Compound A 5 days Data is expressed as mean pg/ml ±standard error of the mean, n = 3-4 in all groups. Significance valuesrelative to vehicle: *p < 0.05; **p < 0.01.

TABLE 2 Pain Reversal by Compound B +/− FK506 in the SNL Rat Model ofAllodynic Neuropathic Pain Drug Dose in SNL Rats % Allodynia Reversal(masked) 24 hr post 7 day post Vehicle (50% DMSO) −1.98 ± 6.90 −7.23 ±6.22 3 mg/kg FK506 BID SC −5.77 ± 3.43 −6.39 ± 4.85 1 mg/kg/day CompoundB    78.50 ± 7.85 **     73.35 ± 11.95 ** osmotic minipump (treat- mentfor 7 days) 1 mg/kg/day Compound B  57.06 ± 14.52*  16.43 ± 19.42osmotic minipump + 3 mg/kg FK506 BID SC (Compound B treated for 7 daysand FK506 treated for 5 days)

TABLE 3 Pain Reversal by Compound C +/− FK506 in the SNL Rat Model ofAllodynic Neuropathic Pain Drug Dose in SNL Rats % Allodynia Reversal(masked) 24 hr post 23 day post Vehicle (50% DMSO) 0.94 ± 5.65   1.72 ±5.08 3 mg/kg FK506 BID SC −0.53 ± 6.21     2.01 ± 5.86 1 mg/kg/dayCompound C TID 66.06 ± 10.71 **  81.48 ± 6.00 ** oral for 5 days 1mg/kg/day Compound C TID  49 ± 13.15* 21.82 ± 4.26  oral for 5 days + 3mg/kg FK506 BID SC for 5 days Data are expressed as mean % MPE, whichrepresents the % allodynia reversal from pre-drug baseline, ± standarderror of the mean, n = 6 in all groups. Significance values vs. vehicle:*p < 0.05; ** p < 0.01.Table 4, below, shows the structures of Compounds A, B and C:

COMPOUND STRUCTURE A

B

C

Studies in Other T Cell Mediated Disease Models

Studies of the mechanism of α_(2B)-receptor agonists mediated effects inChung model of neuropathic pain and in MS-induced pain model indicated asignificant and persistent increase in the number of putative regulatoryT Cells (Tregs) in the lymphatic organs. The inventors exploredα_(2B)-receptor agonists further in the MS model for effects on clinicalcourse of disease and in autoimmune uveitis, endotoxin-induced uveitis,and Dry eye disease. In mice immunized with proteolipid protein, whichcauses a relapsing-remitting form of experimental autoimmuneencephalomyelitis (EAE), treatment with Compound B during thedevelopment of clinical disease has a significant effect on attenuatingsymptoms of MS (FIG. 4). Treatment from days 7-10 reduced the clinicalscore during relapses on days 14 (p<0.01) and 24. Similar results areobtained when mice are treated continuously. In EAE, pro-inflammatoryCD4+ T cells and other inflammatory cells proliferate in the periphery,infiltrate the central nervous system (CNS) which leads to demyelinationcharacterized by a progressive paralysis. Analysis of immune cellinfiltration in the CNS via flow cytometry at the end of the studyrevealed that treatment with Compound B significantly reduced thepresence of immune cells (FIG. 5). This indicates that Compound Bprevented presence of pathogenic T cells in the CNS, resulting inattenuated encephalomyelitis. Compound C also showed similar efficacy inEAE. Following TID oral dosing (3 mg/kg/day) from day 7-13, the clinicalscore was significantly reduced compared to vehicle-treated mice fromdays 13-24 (FIG. 6). Analysis of immune cells in the CNS via flowcytometry on day 37 revealed that Compound C increased the frequency ofregulatory T cells (CD4+CD25hiFoxP3+) in the spinal cord (FIG. 7). Theinventors also performed studies to explore the utility of alpha 2Bagonists in models of ocular T cell mediated inflammation. Experimentalautoimmune uveitis (EAAU) represents an antigen-specific, Tcell-mediated autoimmune response that results in disease in theanterior segment. Compound B, dosed at 1 mg/kg/day from days 1-18 ordays 7-18 following EAAU induction (3 days of oral TID dosing followedby dosing via osmotic minipump), was effective in partially abrogatinganterior inflammation.

Compound B decreased clinical scores of anterior uveitis and decreasedinflammatory cell numbers in the aqueous humor (FIG. 8). In addition,Compound B appeared to be effective in normalizing immune response asseen by decreased neutrophil cell counts in the blood, normalizedlymphocyte populations in the blood and normalized CD4⁺ T cellpopulation in the spleen. A similar effect of Compound B was alsoobserved in the acute endotoxin-induced uveitis (EIU) model. Compound B(1 mg/kg/day delivered by osmotic minipump) significantly inhibitedprotein exudation in the aqueous humor of EIU rats when compared withuntreated or the vehicle-treated (saline) controls (FIG. 9). Theincrease in blood neutrophils and decrease on blood lymphocytepopulations as a result of LPS stimulation was significantly normalizedby Compound B but not the vehicle treatment (FIG. 10). An adoptivetransfer study in a model of dry eye disease shows that the α_(2B)agonist effect on ocular inflammation involves T cells. Following 10days of blower-induced desiccating stress in mice treated with 3mg/kg/day Compound B or vehicle (3 days of oral TID dosing followed bydosing via osmotic minipump), CD4+ T cells were harvested andtransferred to syngeneic nude mice. The recipient mice that received Tcells from Compound B-treated mice (for 10 days during exposure to theblower) had significantly reduced levels of cytokines IL-17 and TNFα,key cytokines that contribute to dry eye disease (FIG. 11). Compound Bwas also tested in a therapeutic mode, wherein the treatment (3mg/kg/day via osmotic minipump) was given to mice that had been exposedto blower previously. In this mode also Compound B significantlyprevented the goblet cell loss and the T cell infiltration into theconjunctiva comparable to Cyclosporine A (FIG. 12). These studiessupport the hypothesis that the mechanism of Compound B, Compound C andother α_(2B) agonists involves immune modulation.

Methods

Spinal Nerve Ligation Model in Rats

The SNL (or Chung) Model in the rat is an accepted standard animal modelof neuropathic pain and is thought to mimic the human causalgiacondition with respect to symptoms (guarding behavior, mechanicalallodynia) and alleviation by pharmacological agents. For instance,morphine does not alleviate the tactile allodynia while Gabapentin (30mg/kg p.o.) results in a 50% alleviation of allodynia. The SNL Model isperformed by tightly ligating the L-5 and L-6 spinal nerves, whichproduces tactile allodynia or sensitivity to light touch as described(Kim and Chung, 1992). Male sprague-dawley rats (100-120 grams; CharlesRiver, Wilmington, Mass.) were anesthetized through inhalation of anisoflurane/oxygen mix. The surgical site was shaved and prepared withbetadine. An incision was made from the thoracic vertebra XIII towarddown the sacrum. The muscle was separated from the spinal vertebra (leftside) at the L4-S2 levels. The L6 vertebra was located and then thetransverse process was carefully removed with a small rongeur tovisually identify the L4-L6 spinal nerves. The L5 and L6 spinal nerveswas isolated and tightly ligated with 6-0 silk thread. A completehomeostasis was confirmed and then the wound was sutured. The durationof the surgery was approximately 20 minutes. A small amount ofantibiotic ointment was applied to the incised area and the animals weretransferred to a plastic recovery cage under a regulatedheat-temperature lamp. Animals were not given any topical or localanesthetics post-operatively because they will inhibit the developmentof the pain syndrome, which is the phenomenon to be studied.

The allodynia is quantitated in the animals having received the Chungsurgery by stimulation with a series of 8 Von Frey hairs on the midplanter area of the surgical hind paw in the up-down manner as describedin the literature (Dixon, 1980). Von Frey hairs are applied in anup-down manner depending on the response until the 50% threshold isestablished. The Von Frey hairs are applied to the plantar surface ofthe surgical paw with just enough force to bend them. A positiveresponse is recorded if the paw is sharply withdrawn. Eight VonFreyhairs were used 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93 and 5.18yielding a gram force of 0.25-15 grams.

Von Frey Analysis:

${\%\mspace{14mu}{Allodynia}\mspace{14mu}{reversal}} = {\left\lbrack \frac{{{Post}\mspace{14mu}{drug}\mspace{14mu}{threshold}} - {{Pre}\mspace{14mu}{drug}\mspace{14mu}{threshold}}}{15 - {{Pre}\mspace{14mu}{drug}\mspace{14mu}{threshold}}} \right\rbrack \times 100}$Mean ± SEM: Mean = average  of  allodynia  reversalsSEM = STDEV/SQ  ROOT  of  n

Testing with compounds is done 2-3 weeks after surgery to establishstable allodynia. In all experimental animals, baseline measurementswere taken prior to drug administration and then at 24 hours and 5-23days post dosing with Alzet osmotic minipump. The investigator wasblinded to the identity of the drug groups. The % allodynia reversal iscalculated as: [(Postdrug threshold−Predrug threshold)/(15−Predrugthreshold)]×100.

Proteolipid-Induced Experimental Autoimmune Encephalomyelitis in Mice

The mouse model of experimental autoimmune encephalomyelitis (EAE) hasbeen used extensively to understand the mechanisms underlying theimmunopathogenesis of MS. Mice immunized with myelin proteins, such asMyelin Binding Protein (MBP), Proteo-Lipid Protein (PLP) and MyelinOligodendrocyte Glycoprotein (MOG) exhibit many similarities to patientswith MS (Friese et al., 2006). Components of the immune system includingT cells, macrophage, and antibodies are important contributors to myelindestruction in EAE mice. Further, inflammation also gives rise tomultifocal regions demyelination culminating in clinical signs ofneurologic dysfunction that include loss of tail tone, abnormal gait andpartial-to-complete hind-limb paralysis. PLP-induced EAE model offersthe ability to simultaneously investigate the pathogenic mechanisms ofCNS inflammation and demyelination and MS-associated pain, amulti-targeted concept that we have optimized here at Allergan. Usingsterile technique proteolipid protein myelin peptide (PLP) (139-159:CHCLGKWLGHPDKFVGITYAL) is mixed 1:1 with incomplete Freund's adjuvant(IFA) (Final concentration of 2 mg/ml PLP). 8-10 week old Female SJLmice (Taconic) are injected subcutaneously with 100 μl PLP/IFA (200 μgPLP/injection) on both the right and left hind-flank (day 0) using a 26G needle. Mice immunized using this protocol experience arelapsing-remitting clinical course with episodes of motor impairmentinterspersed with periods of remission/clinical improvement.Furthermore, these mice display a robust pain phenotype that is mostpronounced during periods of clinical remission.

Using this protocol, the onset of PLP-induced EAE in SJL mice occurs atan incidence of (˜90-100%) and is generally apparent by ˜12 dayspost-immunization, reaching peak disease by 14-21 days. PLP-EAE miceshow physical symptoms of neurologic impairment, progressing frompartial loss of tail tonicity to partial-to-complete hind-limbparalysis. Up to 75% of the mice will experience a relapsing-remittingclinical course with periods of remission flanked by episodes of motorimpairment (hind-limb weakness and paralysis). Histologically, micedisplay extensive inflammation within white matter tracts (areascontaining myelinated axons) of the brain and spinal cord, progressiveinfiltration and accumulation of inflammatory cells, demyelination andaxonal loss. Mice with severe EAE show extensive cellular infiltrates,widespread foci of demyelination. PLP-immunized mice typically displaythe most severe clinical disease during the first episode of neurologicimpairment (days 14-21). In general, the pain phenotype is mostpronounced beginning after remission from the initial demyelinatingevent and is sustained until the mice are euthanized.

Mice were routinely visually scored for behavioral abnormalities on ascale from 0-5; 0—no abnormality, 1—partial loss of tail tonicity(partial limp tail), 2—loss of tail tonicity and hind-limb weakness3—unsteady gait and partial hind-limb paralysis, 4—complete hind-limbparalysis and 5—moribund or dead. Scoring was conducted every-other toevery-third day starting on day 7 post-immunization until the mice weresacrificed. Allodynia was measured by the Von Frey hairs method asdescribed earlier. At the end of the study spleen, cervical lymph node,spinal cord and brain were collected to perform flow cytometry.

Dry, Desiccating Stress (DS) in Mice

C57BL/6 (C57BL/6NTac) and B6.Cg/NTac-Foxn1nuNE9 were purchased fromTaconic, Inc. (Germantown, N.Y.). Mice were used at 6-10 weeks of age.Animal studies approval was obtained from the Allergan Animal Care andUse Committee. All studies adhered to the Association for Research inVision and Ophthalmology statement for the Use of Animals in Ophthalmicand Vision Research. As described in the literature dry eye was inducedby treating mice with subcutaneous injections of scopolaminehydrobromide (0.5 mg/0.2 ml; Sigma-Aldrich, St. Louis, Mo.) three timesa day alternating between the left and right flanks (Neiderkorn et al,2006). Mice were placed in a cage containing perforated plastic screenson each side of the cage to permit airflow from fans (one fan on eachside of the cage) for 16 hr/day in a hood (AirClean Systems, Raleigh,N.C.). Room humidity was kept below 40%. Desiccating stress (DS) wasinduced for 10 consecutive days. Spleens and cervical lymph nodes (CLN)were collected from mice subjected to DS and NS, and onedonor-equivalent of either spleen or CLN CD4+ cells was transferred i.p.to syngeneic nude mice. One splenic equivalent of T cells was equal to5×10⁷ cells. Three days later samples were collected for analysis. Fortear collection, 1.5 μL of PBS was placed on each eye, and then 1 μL oftear was collected from both eyes and placed in 8 μL of cytokine assaybuffer (Beadlyte; Millipore, Billerica, Mass.). Buffer and tear fluidwere collected by capillary action using a 1 μL volume glass capillarytube (Drummond Scientific, Broomhall, Pa.) that was placed in the tearmeniscus of the lateral canthus. Samples were frozen at −80° C. untilthe time of assay. Histological analysis was done by staining Lacrimalgland samples with antibody to CD4 and H&E staining to quantify gobletcells and T cells in the conjunctiva. To test the compounds in atherapeutic mode, the animals were exposed to the desiccating stress fortwo weeks and then allowed to rest for 7 days in regular housing cages.The animals were then re-exposed to desiccating stress for an additional7 days to mimic relapsing form of chronic dry eye disease. Drugtreatment with Compound B was started 2 days before re-exposure todesiccating stress. In both the studies Compound B was dosed at 3mg/kg/day. Adoptive transfer, tear cytokine analysis and histologicalanalysis were performed as described above.

Experimental Autoimmune Anterior Uveoretinitis in Rat

Male Lewis rats (180-200 g) were immunized by a single left hind-footpadinjection with 150 μg (in 100 μL) of purified MAA complex from bovineeye. MAA proteins were suspended in phosphate-buffered saline (PBS,pH7.2) and emulsified (1:1 v/v) in complete Freund's adjuvant (CFA, VWRScientific) containing 1 μg/100 μl of Pertussis Toxin (PTx) emulsionmixture. Control animals were injected with PBS emulsified with CFA andPTx.

Assessment of intraocular inflammation was conducted starting day 7after immunization, Animals were examined every other day between day 7and 19 post-immunization for clinical signs and symptoms of uveitisusing slit lamp microscopy. Aqueous humor was collected to evaluate thenumber of inflammatory cells and protein levels. Cell counts were donewith 10 ul with a hemacytometer under the light microscope. Proteinconcentration was measured with a Protein assay solution from Bio-Rad,using BSA as the protein standard. Pro-inflammatory cytokine andchemokine levels were measured on Luminex (Biosource-Invitrogen,Carlsbad, Calif.). Blood, spleen, and eyes were harvested on days 11,14, and 19 to determine blood leukocyte differentiation, splenic T cellactivation status via Flow cytometry, as well as histopathology.

Endotoxin-Induced Uveitis in Rats:

Female Lewis rats (180-200 grams) were purchased from Charles RiverLaboratory. Rats were footpad-injected (hind left side) with either 100μl of 1 mg/ml LPS (Sigma) solution (in sterile pyrogen-free saline) or150 μl of sterile pyrogen-free saline. Animals were sacrificed at 24hours following LPS injection. Aqueous humor was collected and analyzedto determine inflammatory cell counts, levels of cytokines and chemokineconcentration as well as total protein concentrations. Blood leukocytedifferentiation and histopathology were also determined if required.

Formulations:

Compound A was formulated in 50% DMSO (Sigma, St. Louis, Mo.). Thissolution was loaded into the osmotic minipumps (Model 1007D, AlzetCorp., Palo Alto, Calif.) set to deliver the drug at a rate of 0.5 ul/hrresulting in a final dose of 2.4 mg/kg/day. The vehicle for thesestudies is 50% DMSO administered via osmotic minipumps at a rate of 0.5ul/hr/kg.

Compound B was formulated in 50% DMSO (dimethyl sulfoxide; Sigma, St.Louis, Mo.). This solution was dosed orally TID at a dose of 0.3 or 1mg/kg. For dosing in the osmotic minipumps (Model 1007D, Alzet Corp.,Palo Alto, Calif.) the compound was loaded onto the pumps set to delivera final dose of either 1 or 3 mg/kg/day. The vehicle for these studiesis 50% DMSO.

Compound C was formulated first in 100% DMSO (Sigma, St. Louis, Mo.),then diluted down in 30% DMSO for a 10 mg/ml solution; 15% DMSO for a 3mg/ml solution and further dilutions were made in water. The compoundwas dosed orally TID at a dose of either 0.3 or 1 mg/kg for a totaldaily dose of 1 or 3 mg/kg/day, respectively.

Flow Cytometry

Superficial cervical lymph node cells and spleen cells were obtained bygentle mechanical processing. CD4⁺ cells were isolated by using a CD4+isolation column (Miltenyi Biotech, Auburn, Calif.) according to themanufacturer's protocol. The brain and spinal cord were mechanicallydisaggregated and mononuclear cells of the CNS were isolated using 37.5%Percoll (Sigma-Aldrich).

To determine surface expression of CD4 (Helper T cells), CD8 (killer Tcells), CD25 (activated helper T cells and regulatory T cells), CD45(macrophage and microglial cells) and F4/80 (macrophage and microglialcells) 5×10⁵ cells/100 μL FACS buffer (PBS, 0.02% sodium azide[Sigma-Aldrich] and 2% bovine serum albumin) were incubated withappropriate antibodies from BD biosciences, Mountain View, Calif.Isotype control antibodies used for each of the antibodies. The cellswere washed two times in FACS buffer and resuspended at 5×10⁵ cells/100μL buffer. The tubes containing biotin-labeled antibody received 1.5 μLof an accessory staining pigment (Streptavidin PerCP; BD-Pharmingen) andwere placed on ice for 20 minutes in the dark. Expression was analyzed(FACSCalibur with CellQuest software; BD Biosciences, Mountain View,Calif.).

Luminex Analysis of Cytokines

The levels of cytokines were measured with a sensitive, fluorescentmultiplex immunobead assay (Luminex; Biosource-Invitrogen, Carlsbad,Calif.), 9 plex rat cytokine/chemokine (RCYTO-80K-09) panel fromMillipore was used. Cytokine levels in samples were analyzed by usingthe corresponding Millipore cytokine Beadmate pairs. For the Luminexassay, a 96-well filter plate (Millipore) was pre-wetted with 25 μl ofBeadlyte cytokine assay buffer. A vacuum manifold (Millipore, Billerica,Mass.) was used to aspirate the buffer from the wells. 25 μl of samplewas placed in each well. The beads (25 μl) were pipetted into the wells.Standard curves for each cytokine were generated in duplicate by placing25 μl of the appropriate dilution of standards purchased from Millipore.The plate was incubated overnight with gentle shaking in the dark at 4°C. Beads were washed with Beadlyte cytokine assay buffer (Millipore) andwash buffer was eliminated using a vacuum manifold. 25 μl of theappropriate biotin-conjugated secondary antibody (Millipore) was addedto each well for 90 minutes at room temperature with gentle shaking.Beads were incubated with Beadlyte streptavidin-phycoerythrin (1:25dilution in Beadlyte assay buffer) for 30 minutes at room temperaturewith gentle shaking. Beads were washed, resuspended in 125 μl ofBeadlyte buffer, and analyzed by Luminex 100 instrument (LuminexCorporation, Austin, Tex.). The mean fluorescence intensities obtainedfrom 50 beads per cytokine minimum were analyzed using Upstate Beadviewsoftware. Standard curves were generated (8 data points including a zerostandard run in duplicate) using a four- or five-parametric logisticcurve. R-squared values were between 0.99 and 1. Data are expressed inpg/ml or ng/ml values.

What is claimed:
 1. A method of treating dry eye disease in a patient inneed of such treatment, the method comprising administering to thepatient an alpha-2B receptor agonist, wherein the alpha-2B receptoragonist is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R and R¹ areindependently halogen or alkyl; R² is H or alkyl which may beunsubstituted or substituted with a heteroaryl or aryl; and Het isimidazolinyl.
 2. The method of claim 1, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein the agonist is administered for an initial period, and thenadministered for a second period after a withdrawal period has elapsed.4. The method of claim 3, wherein each of the initial, second, andwithdrawal periods is independently one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen days, orone, two, three, or four weeks.
 5. The method of claim 1, wherein theagonist is administered at a therapeutically effective dose.
 6. Themethod of claim 2, wherein the agonist is administered for an initialperiod, and then administered for a second period after a withdrawalperiod has elapsed.
 7. The method of claim 6, wherein each of theinitial, second, and withdrawal periods is independently one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, or fourteen days, or one, two, three, or four weeks.
 8. Themethod of claim 2, wherein the agonist is administered at atherapeutically effective dose.